Compositions of digestive enzymes and salts of bile acids and process for preparation thereof

ABSTRACT

Disclosed are gastric acid-resistant polymer-coated digestive enzymes/ursodeoxycholate compositions, process for their preparations and methods of treating digestive disorders, impaired liver function, cystic fibrosis, regulating the absorption of dietary cholesterol, and for dissolving gallstones by administering the compositions to a mammal in need of such treatment.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to digestive enzymes and salts of bile acids, andmore particularly salts of ursodeoxycholic acid compositions foringestion by a mammal, a process for preparing said compositions, and amethod for treating digestive disorders, impaired liver function, cysticfibrosis, regulating dietary cholesterol absorption and for dissolvinggallstones by administering said compositions to a mammal in need ofsuch treatment.

2. Reported Developments

It is known in the prior art that pancreatic enzymes administered tomammals can remedy enzyme deficiency caused by various diseasedconditions of the pancreas, such as cystic fibrosis, pancreatitis,pancreatic enzyme deficiency and old age. Oral administration ofcompositions containing these enzymes requires the presence of certainconditions in order for them to be safe and effective as will bedescribed hereunder.

Pancreatic enzymes produced by the patient's pancreas are released intothe duodenum, the pH of which is close to neutral or slightly alkaline.Under these pH conditions the enzymes are active and digestion of thefood by the enzymes proceeds normally in the upper segment of theintestine. However, when pancreatic enzymes are administered exogenouslyto the patient, the gastric conditions in the stomach, namely thepresence of acid and pepsin, will irreversibly inactive the enzymes.Therefore, orally administered enzymes must be protected against gastricinactivation so that they remain intact during their transit through thestomach into the duodenum.

Once the exogenously introduced enzymes reach the duodenum, anotherrequirement must be satisfied: the enzymes must be released from theirprotective environment and intimately mixed with the food transferredfrom the stomach to effect digestion.

U.S. Pat. No. 4,079,125 incorporated herein by reference, addressesthese requirements in a composition containing these enzymes andprovides preparative methods for making the compositions. Thecompositions provided by said patent comprise: an enzyme concentrate ina binder selected from the group consisting of polyvinylpyrrolidone,microcrystalline cellulose, cellulose acetate phthalate, methylcelluloseand alginic acid; a stabilizer selected from the group consisting ofcalcium carbonate, polyvinylpyrrolidone, cellulose acetate phthalate,methylcellulose, starch and modified starches and alginic acid; adisintegrant selected from the group consisting of citric acid, sodiumcarbonate, sodium bicarbonate, calcium carbonate, starch and modifiedstarches and alginic acid; said mixture is coated with a non-porous,pharmaceutically acceptable enteric coating polymer which is insolublein the pH range of from about 1.5 to about 5 normally present in gastricfluids, and soluble at a pH of from about 6 to about 9, the normal pHrange for mammalian intestinal fluids.

The orally administered composition passes through the stomach whilebeing protected against the acidic environment by its acid-insolublecoating which then disintegrates in the neutral to basic environment ofthe upper intestine releasing the enzymes from the composition. Theprocess of making the compositions includes the provision of using asolvent and avoiding the presence of water in the blending step of theenzyme/binder/disintegrant, since it is believed that water deactivatessome of the enzymes.

Contrary to the teaching of U.S. Pat. No. 4,079,125, it has now beendiscovered that the complete exclusion of the water (anhydrouscondition) during the process of preparing the enzymes/salts ofursodeoxycholic acid compositions in the form of microtablets andmicrospheres, leads to products that are extremely friable, tend tocrumble into pieces upon drying in a fluidized bed dryer or conventionalcoating pan and disintegrates upon initiation of the polymer coatingstep. This results in large amounts of dust and agglomeration of thebeads into multiplets during the process as well as improper doses ofthe enzymes upon administration to the patient when quality controlfails to adequately sort-out and discard rejects.

It is also known that ursodeoxycholic acid (hereinafter UDCA or bileacid) is capable of augmenting liver function, dissolving gallstones andimproving the nutritional state of patients having cystic fibrosiscaused by hepatobiliary complications. See for example, UrsodeoxycholicAcid Dissolution of Gallstones in Cystic Fibrosis, Sahl, B., Howat, J.,Webb, K., Thorax, 43:490-1 (1988); Effects of Ursodeoxycholic AcidTherapy for Liver Disease Associated with Cystic Fibrosis, Colombo, C.,Setchell, K. D., Podda, M., Crosignani, A., Roda A., Curcio, L., Ronchi,M. and Giunta, A., The Journal of Pediatrics, 117:482-489 (1990);Effects of Ursodeoxycholic Acid Treatment on Nutrition and LiverFunction in Patients with Cystic Fibrosis and Longstanding Cholestasis.Cotting, J., Lentze, M. J. and Reichen, J., Gut 31:918-921 (1990). Also,UDCA has recently gained acceptance as an effective therapeutic modalityto dissolve small to medium size cholesterol gallstones in gallstoneafflicted patients. See for example, The Effect of High and Low Doses ofUrsodeoxycholic Acid on Gallstone Dissolution in Humans, Salen, G.,Colalillo, A., Verga, D., Bagan, E., Tint, G. S. and Shefer, S.,Gastro., 78:1412-1418 (1980); Ursodeoxycholic Acid: A Clinical Trial ofa Safe and Effective Agent for Dissolving Cholesterol Gallstones, Tint,G. S., Salen, G., Colalillo, A., Graber, D., berga, D. Speck, J. andShefer, S., Annals of Internal Medicine, 91:1007-1018 (1986); ClinicalPerspective on the Treatment of Gallstones with Ursodeoxycholic Acid,Salen, G., J. Clin. Gastroenterology, 10 (Suppl. 2):S12-17 (1988);Nonsurgical Treatment of Gallstones, Salen, G. and Tint, G. S., NewEngland J. Med., 320:665-66 (1989); and Reducing Cholesterol Levels,Weigand, A. H., U.S. Pat. No. 3,859,437. The recommended dosage is 10 to15 mg/kg of body weight. In some pateints muchj higher dosages (forexample, about 30 mg/kg of body weight) are required to achieve limitedbenefits. However, in some patients undesirable side effects (such assevere diarrhea) seriously limit the use of this drug. The reasons forthis wide variation of dosage requirements for therapeutic effectivenessand associated side effects are not completely understood. Onehypothesis is that the free acidic form of UDCA is only partiallyneutralized in the upper intestine to its sodium salt form. The residualfree acidic form of UDCA is poorly absorbed from the intestine, and agood portion of the administered dosage is excreted intact with feces.When a higher dosage of the acidic form of UDCA is administered to thepatient, a large portion of it is neutralized in the distal parts of theintestine which in turn induces diarrhea, a highly undesirable sideeffect. Also, if the acidic form of UDCA is to be converted into itssalt form in the duodenum, it will temporarily exhaust the bufferingcapacity of the duodenum and it will render the upper intestinepartially acidic. The acidic pH impedes the function of the pancreaticenzymes and UDCA cannot emulsify fats and facilitate the hydrolysis oflipids. Furthermore, the many therapeutic benefits derived from the saltforms of UDCA cannot be realized. It should then follow, accordingly,that the salt forms of UDCA should be administered to patients in needof UDCA. U.S. Pat. No. 3,859,437 recommends the administration of a"small but effective amount sufficient to effect a reduction in thecholesterol level of said human being of the compound3α7β-dihydroxy-5β-cholanic acid (UDCA) and the non-toxicpharmaceutically acceptable salts thereof". However, administering thesalt form of UDCA to patients has no advantage over the acidic form ofUDCA and does not accomplish the desired results since the salt form ofUDCA is converted back to the insoluble acidic form of UDCA by gastricacidity. Furthermore, the salt forms, i.e., sodium or potassium, of UDCAare extremely bitter-tasting, and in most patients cause esophagealreflux, nausea and vomiting. Because of these highly undesirableorganoleptic and gastric side effects, the salt forms of UDCA has notgained therapeutic utility in the treatment of biliary diseases.

Pancreatic enzymes and salts of UDCA complement one another in thedigestive system of a mammal. A dietary supplement containing both theenzymes and salts of UDCA would provide in a convenient pre-determineddose the remedy needed to treat the above-described diseased states.However, the acidic form of UDCA is incompatible with pancreaticenzymes. Pancreatic enzymes/UDCA compositions have a pH of about 5 to5.5. Under these acidic conditions most pancreatic enzymes show a lowbiological activity of about 10% to 40%. Lipase is especially affectedby the low pH for the reasons that: UDCA is only sparingly soluble inaqueous media and is inefficient to emulsify fats; and the acidic UDCAinactivates lipase since lipase requires a basic pH for biologicalactivity.

Pancreatic enzymes/UDCA containing compositions also lack sufficientshelf-life due to the denaturing and detergent effects of UDCA on thepancreatic enzymes. Because of these incompatibilities between UDCA andpancreatic enzymes the many benefits derivable from their combinationscould not be realized by the prior art.

It has now been discovered that the problems associated individuallywith enteric coated microtablets and microspheres containing pancreaticenzymes and compositions containing UDCA, may be overcome in a dietarysupplement containing both the pancreatic enzymes and a salt of UDCA. Inaccordance with the discovery, UDCA is first converted to apharmaceutically acceptable salt, such as the sodium or potassium saltand then used in a combination with pancreatic enzymes in a composition.Such salts are highly effective to emulsify fats and lipids at a basicpH and facilitate the hydrolysis of the emulsified fat globules. As aresult, fat digestion is greatly enhanced. The salts are also moreeffective than the insoluble acidic form of UDCA to lyse mucus whichblocks the intestinal surfaces and prevents absorption of metabolitesthat results in poor nutrition in cystic fibrosis children.

Pancreatic enzymes then are combined with a salt of UDCA and bufferedwith a biologically compatible, pharmaceutically acceptable buffer thatprevents deactivation of the enzymes and preserves the naturalbiological activities of both the enzymes and the salt of UDCA. Thepancreatic enzymes/bile salt composition thereby resulting in productsthat do not crumble upon drying or disintegrate upon initiation of thepolymer coating procedure. The bitter taste and associated gastricdisadvantages of UDCA salts are also eliminated by the polymer coatingwhich prevents solubilization of the product in the mouth and stomach ofthe patient.

Still further, it has been discovered that microspheres in the range of10 to 80 mesh size (about 2.0 to 0.177 mm range) can be preparedutilizing bile salts as seeds to build up the microspheres. Such smallparticle size microspheres are especially beneficial for use to treatpancreatic enzymes/bile salt deficiencies in cystic fibrosis children.

SUMMARY OF THE INVENTION

The invention will be described with particular reference to salts ofursodeoxycholic acid, however, it is to be understood that salts ofother bile acids and salt complexes thereof including their isomers maybe used as well.

In accordance with the present invention, digestive enzymes/salt of UDCAcompositions are provided which possess desirable characteristicsheretofore absent in proposed or existing prior art products.

The digestive enzymes/salt of UDCA is instantly soluble in water, whileUDCA alone on in combination with a digestive enzyme is essentiallyinsoluble.

Only the ionized or salt form of UDCA or the conjugated derivatives ofUDCA are absorbed from the intestine, while the acidic form of UDCA isinsoluble and passes through the intestine intact, unless it isconverted to the sodium salt by the intestinal buffers. However, manypatients, such as patients with cystic fibrosis, pancreatitis, BillrothI & II diseases and some elderly people, are partially deficient inbicarbonate secretion and lack neutralization capacity to convert theacidic form of UDCA to the sodium salt of UDCA. These patients will onlypartially benefit from UDCA therapy. The salt of UDCA-containingcomposition of the present invention overcomes this problem by beinginstantly soluble in the intestinal juices and absorbable from theintestine. Additionally, the composition also provides extra bufferingcapacity to neutralize the acid chyme that is present in the intestineand greatly facilitates the efficient digestion of fats and lipids inthe upper intestine.

The digestive enzymes/salt of UDCA composition is microencapsulated andcoated with an acid-resistant polymer-coating, which protects thecomposition from gastric acid and from conversion of the salt of UDCA tothe acidic form of UDCA. The polymer-coated microcapsules are tastelessand the problem associated with the offensive bitter taste of theuncoated acidic form or the uncoated salt of UDCA is thereby alleviated.

The microcapsules uniformly disperse with the food in the stomach anddeliver high levels of biologically active digestive enzymes/salts ofUDCA into the duodenum. Once in the duodenum, the polymer coatingdissolves within about 10 to 30 minutes and the enzymes/salts of UDCAare released to enhance digestion of fats and lipids. As a result, thenatural digestive conditions in the intestine are re-established.Epigastric pain, cramps, bloating, flatulence and stool frequencyassociated with maldigestion of fatty foods are reduced.

Soluble salts of UDCA and conjugated derivatives of UDCA are absorbedmore efficiently and in a greater quantity from the intestine than theinsoluble acidic form of UDCA, resulting in a more efficient stimulationof the liver enzymes to conjugate ursodiol (UDCA). The increasedconcentration of the conjugated ursodiol stimulates bile flow, enhancesthe displacement of toxic bile acid metabolites from the hepatocytes,decreases cholesterol secretion into bile, alters thecholesterol/phospholipid ratio of secreted bile and decreases theabsorption of dietary cholesterol from the intestine. The overall resultis decreased biliary cholesterol saturation, increased bile flow,dissolution of already formed cholesterol gallstones and protection ofthe liver from accumulated toxic metabolites.

The digestive enzymes/salt of UDCA composition for the treatment ofenzyme/UDCA deficient mammals comprises a blend of ingredients and acoating expressed in weight per weight percentages based on the totalweight of the composition:

a) from about 71 to about 90.0% of a concentrate of an enzyme selectedfrom the group consisting of pancreatic proteases, lipases, nucleasesand amylases;

b) from about 0.3 to about 13% of a UDCA salt in powder or microsphereform;

c) from about 0.8 to about 5% of a buffering agent selected from thegroup consisting of about 0.25 to about 5.0% sodium carbonate (anhydrouspowder), sodium bicarbonate, potassium carbonate, potassium bicarbonateand ammonium carbonate and from about 0.25 to about 1.5% tromethamine,diethanolamine and triethanolamine;

d) from about 0.5 to about 8% of a disintegrant selected from the groupconsisting of starch and modified starches, microcrystalline celluloseand propylene glycol alginate;

e) from about 0.3 to about 10% of an adhesive polymer selected from thegroup consisting of polyvinylpyrrolidone, microcrystalline cellulose,hydroxypropyl cellulose, cellulose acetate phthalate and a 60:40 blendof methyl cellulose and hydroxypropyl methyl cellulose; and

f) from about 7.0 to about 15% of a non-porous, pharmaceuticallyacceptable gastric acid-resistant polymer-coating which contains lessthan 2% talc and which is insoluble in the pH range of from about 1.5 toabout 5 but is soluble in the pH range of from about 5.5 to about 9.

The digestive enzymes of the present invention includes Pancreatin ofmultiple strength, Pancrelipase, Trypsin, Chymotrypsin, Chymotrypsin B,Pancreatopeptidase, Carboxypeptidase A, Carboxypeptidase B, GlycerolEster Hydrolase, Phospholipase A2, Sterol Ester Hydrolase, Ribonuclease,Deoxyribonuclease, α-Amylase, Papain, Chymopapain, Bromelain, Ficin,β-Amylase, Cellulase and β-Galactosidase (Lactase).

The salts of UDCA includes sodium, potassium, ammonium, tromethamine,ethanolamine, diethanolamine and triethanolamine salts or saltcomplexes.

In accordance with the present invention, the enzyme/bile saltcomposition is prepared by a process comprising the steps of:

a) blending dry, powdery ingredients selected from the group consistingof (i) from about 71 to about 90% w/w of an enzyme from the groupconsisting of pancreatic proteases, lipases, nucleases and amylases;(ii) from about 1.0 to about 13% w/w of a salt or salt complexes of UDCAselected from the group consisting of sodium, potassium, ammonium,tromethamine, ethanolamine, diethanolamine and triethanolamine; (iii) abuffering agent selected from the group consisting of from about 0.25 toabout 5.0% w/w sodium carbonate (anhydrous), sodium bicarbonate,potassium carbonate, potassium bicarbonate and ammonium carbonate, andfrom about 0.25 to about 1.5% w/w, tromethamine, diethanolamine andtriethanolamine; (iv) of from about 0.5 to about 8.0% w/w a disintegrantselected from the group consisting of starch and modified starches andmicrocrystalline cellulose and propylene glycol alginate; and (v) fromabout 0.3% to about 10% w/w of an adhesive polymer selected from thegroup consisting of polyvinylpyrrolidone, cellulose acetate phthalate,hydroxypropyl cellulose and methylcellulose;

b) wetting said blended ingredients with a liquid to cause the blend tostick together, wherein said liquid is selected from the groupconsisting of: 1%-25% w/w ethanol/75%-99% w/w 2-propanol/0.2%-2% w/wwater; 98%-99% w/w 2-propanol/0.2%-2% w/w water, 1%-25% w/wmethanol/0.2%-2% w/w water/75%-98% w/w 2 propanol/1%-5% w/wethylacetate;

c) granulating or extruding the liquid-wetted blend through a 10 to a 18mesh S/S screen;

d) converting the granules to a uniform diameter particle size;

e) compacting the uniform particles to spherical particles;

f) drying the spherical particles;

g) separating the spherical particles if not of uniform size accordingto desired sizes using U.S. Standard sieve screens;

h) coating the particles with a gastric acid-resistant polymer thatdissolves under neutral or slightly basic conditions; and

i) drying the polymer-coated spherical particles.

DETAILED DESCRIPTION OF THE INVENTION

In preparing the pancreatic enzymes/bile salt containing microspheres ofthe present invention utilizing the extrusion, uni-sizer andmarumerization process (later described) moisture must be included inthe liquid or solvent-adhesive composition to render the adhesivepolymer sticky enough to bind the enzymes/bile salt-containing fluffypowder into a pliable, solid mass. This prevents the crumbling of themicrospheres during the drying and coating steps as well as allows thepreparation of much smaller particle size microspheres, i.e. in therange of 10 to 80 mesh. Accordingly, it was found that the moisturelevel during the preparation of the composition should be in the rangeof from about 0.2% w/w to about 2.5% w/w, preferably, in the range of0.2% w/w to 1.5% w/w, and most preferably in the range of 0.2% w/w to1.0% w/w. When the compositions contained such amounts of moisture, themicrospheres were found to be stable on aging and biological activitywas preseved as long as the moisture level did not exceed about 2.5% w/wof the total composition.

Further reference is now made to the process of preparing compositionsof the present invention.

The process for the manufacture of microspheres consists of:

1) Blending the dry, powdery ingredients together in a conventionalblender and wetting the composition with a suitable liquid composition,hereinbefore described, that causes the dry blend to stick together. Thestickiness of the blend can be tested by compressing a handful of theblend in the palm of the hand. If the composition is compressible andsticks together but readily crumbles when squeezed between the fingers,sufficient liquid has been added to the composition for processing inthe subsequent granulation step.

2) Granulating or extruding the liquid moistened composition through a10 or a 18 mesh S/S screen using an oscillating/reciprocating granulatoror a twin-screw extruder at a medium-to-high speed.

3) Classifying the granulated particles in a so-called "uni-sizervessel" that rotates at 15 to 45 rpm for about 5 to 10 minutes. (Theparticles in the "uni-sizer vessel" are converted to a uniform diameterparticle size.)

4) Compacting the uniform particles in a marumerizer, (a cylindricalvessel with a rotating disk at the bottom) for about 15 to 70 seconds.An alternative method of compacting the microspheres can also beachieved in a rotating conventional coating pan. In this case, theparticles are tumbled in the pan for about 15 to 30 minutes,occasionally wetting the particles with a fine mist of the liquidcomposition.

5) Drying the spherical particles in an oven under a stream of warm anddry air not exceeding 35° C. and 40% relative humidity.

6) Separating the microspheres according to the desired sizes using U.S.Standard sieve screens.

7) Coating the desired and classified microspheres (for example, in the16 to 20 mesh and separately in the 30 to 60 mesh size range) with anacid-resistant polymer in fluidized bed coating equipment, or in aconventional coating pan according to standard operating procedures asdescribed in the manufacturer's instruction manual.

8) Drying the polymer coated cicrospheres in an oven under a stream ofwarm and dry air not exceeding 35° C. and 40% relative humidity untilall the volatile substance (moisture and solvents) are removed.

The following examples will further serve to illustrate the compositionsof the present invention wherein the compositions and the process ofpreparing them will be described with reference to microsphere forms;however, it is to be noted that the microtablet form of the compositionand the process of making it is also intended to be covered by thepresent invention. The process of making the microtablet form of thecomposition is analogous to that of making the microspheres with theexception that the 40 to 80 mesh particles are compressed together intomicrotablets of 0.5 mm to 2.5 mm with a suitable tablet press andpolymer coated, and should be understood by those skilled in the art.

EXAMPLE I Generalized Formula Compositions (polymer coated)

    ______________________________________                                        Ingredients         % w/w                                                     ______________________________________                                        Disintegrant        0.9-16%                                                   Salt of Bile acid   0.3-13%                                                   Buffering agent (anhydrous)                                                                       0.8-5%                                                    Enzymes             90.0-71%                                                  Adhesive Polymer    0.3-19%                                                   Polymer coat/talc mixture                                                                         7.0-15%                                                   ______________________________________                                    

EXAMPLE II Formula Composition

    ______________________________________                                                         IIA (uncoated)                                                                             IIB (coated)                                    Ingredients      % w/w        % w/w                                           ______________________________________                                        Disintegrant     3.0          2.7                                             Sodium Ursodeoxycholic acid                                                                    5.3          4.7                                             Buffering agent (anhydrous)                                                                    1.0          0.9                                             Enzymes          89.3         79.7                                            Adhesive Polymer 1.4          1.3                                             Polymer coat/talc mixture     10.7                                            ______________________________________                                    

EXAMPLE III Formula Composition

    ______________________________________                                                         IIIA (uncoated)                                                                            IIIB (coated)                                   Ingredients      % w/w        % w/w                                           ______________________________________                                        Disintegrant     3.0          2.7                                             Sodium-Ursodeoxycholic acid                                                                    5.3          4.7                                             Buffering agent (anhydrous)                                                                    1.0          0.9                                             Enzymes          89.3         79.7                                            Adhesive Polymer 1.4          1.3                                             Polymer coat/talc mixture     10.7                                            ______________________________________                                    

EXAMPLE IV Formula Composition

    ______________________________________                                                          IVA (uncoated)                                                                            IVB (coated)                                    Ingredients       % w/w       % w/w                                           ______________________________________                                        Disintegrant      2.0         1.8                                             Potassium-Ursodeoxycholic acid                                                                  1.5         1.4                                             Buffering agent (anhydrous)                                                                     2.0         1.8                                             Enzymes           90.5        83.1                                            Adhesive Polymer  4.0         3.6                                             Polymer coat/talc mixture     8.3                                             ______________________________________                                    

EXAMPLE V Pancreatic Enzyme/Bile Salt Composition

    ______________________________________                                        Ingredients           % w/w                                                   ______________________________________                                        Bile salt starting seed (20-40 mesh)                                                                12.8%                                                   Disintegrant          2.3%                                                    Buffering agent (anhydrous)                                                                         2.1%                                                    Enzymes               65.0%                                                   Adhesive polymer mixture                                                                            7.1                                                     Polymer coat/talc mixture                                                                           10.7                                                    ______________________________________                                    

The bile salt starting seeds in Example V were prepared as outlined inExample VII. Suitable bile acids, bile salts and bile acid esters toprepare starting seeds in the particle size range of 20-60 mesh are:Ursodeoxycholic acid; sodium, potassium and ammonium salts ofursodeoxycholic acid; ethyl and propyl esters of ursodeoxycholic acid;glycyl and tauroursodeoxycholic acid; sodium, potassium and ammoniumsalts of glycyl and tauroursodeoxycholate; N-methyl glycylursodeoxycholate and N-methyl tauroursodeoxycholate.

                  TABLE I                                                         ______________________________________                                        Distribution of the Microspheres According to Sizes                                            Example IIB   Example IIIB                                   Mesh Size                                                                              (mm)    Microspheres (%)                                                                            Microspheres (%)                               ______________________________________                                        10       2.00    --            3.5                                            20       0.84    10.0          57.0                                           40       0.42    53.8          32.7                                           60       0.25    28.6          5.2                                            80        0.177   7.6          1.6                                            ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        Moisture Content & Stability of the Microspheres                                     Moisture Stability                                                                              Moisture   Stability                                        Content (%)                                                                            (4 mo.)* content (%)                                                                              (4 mo.)*                                  Mesh Size                                                                              IIB             IIIB                                                 ______________________________________                                        20       1.1        99%      1.6      98%                                     40       0.9        98%      1.9      96%                                     60       0.8        100%     2.5      95%                                     80       0.9        98%      2.7      85%                                     ______________________________________                                         *Lipase, Amylase and Protease Activities assayed according to USP XXII.  

EXAMPLE VI Preparation of Salts of Ursodeoxycholic Acid

In general, UDCA was converted to the sodium or potassium salt (Na-UDCA,K-UDCA) by dissolving UDCA in a suitable solvent and titrated with awater soluble alkaline hydroxide, carbonate or bicarbonate solution(e.g. sodium hydroxide, sodium carbonate, sodium bicarbonate, potassiumhydroxide, potassium carbonate, potassium bicarbonate solutions, etc.)until the pH has reached pH 8.6. The solvent was removed by evaporationor by distillation and the UDCA-salt was recovered from the media byspray drying or by lyophilizing the remaining solution.

In a more detailed process, 20 g of UDCA was dissolved in 100 ml ofalcohol (methanol, ethanol, isopropanol or an other suitable alcoholthat was easily removed after UDCA has been neutralized) and a 10%-30%solution of hydroxide, bicarbonate or carbonate solution of Na, K, etc.was added to the reaction mixture, with rigorous mixing. The UDCAsolution was titrated until the pH reached 8.6. The alcohol was removedfrom the reaction mixture on a rotary evaporator, and the aqueoussolution was processed to recover the solid Na-UDCA by lyophilization orby spray-drying.

In a modified procedure, a cation exchange resin in the sodium form(AMBERLITE IRC-72) was used to prepare the Na-UDCA. The resin wassuspended in methanol. The methanol/cation exchange resin was pouredinto a 4 cm×40 cm column and washed with 500 ml of methanol to removetraces of water. A 5% solution of UDCA in methanol was passed throughthe resin, followed by washing the column with 200 ml of methanol. Theeluent was collected, the methanol evaporated and the Na-UDCA wascollected as fine crystals.

EXAMPLE VII Preparation of Bile Salt Starting Seeds

    ______________________________________                                                       % w/w                                                          ______________________________________                                        Bile Salt        60.7                                                         Disintegrant     16.0                                                         Buffering agent   4.6                                                         Adhesive polymer 18.7                                                         ______________________________________                                    

The process of making the bile salt-containing starting seeds consistedof: 1) blending the bile salt, disintegrant and the buffering agenttogether for 10-minutes; 2) spraying the composition with the adhesivepolymer mixture until the powdery blend agglomerated; and 3) granulatingor extruding the liquid moistened composition through a 10 or 18 meshS/S screen using an oscillating/reciprocating granulator or a twin-screwextruder. The subsequent processing steps were the same as outlined inSteps (3) through (6) in the "Detailed Description of the Invention".

Referring to ingredients used in the above examples:

Suitable Disintegrant in Examples I through V and Example VII are:Explotab (Mendell, Inc.), microcrystalline cellulose, and propyleneglycol alginate (Kelco Co.)

Suitable Buffering Agents in Examples I through V and Example VII are:sodium carbonate (anhydrous), sodium bicarbonate, potassium carbonate,potassium bicarbonate, ammonium carbonate, tromethamine, diethanolamineand triethanolamine.

Suitable Enzymes in Examples I through V and Example VII are:Pancreatin, Pancrelipase and Pancreatin concentrates of high potency.

Suitable Bile Salts in Examples I through V and Example VII are: sodiumand potassium salts of ursodeoxycholate, glycyl and taurineursodeoxycholate, N-methyl glycyl and N-methyl tauroursodeoxycholate,and organic complexes of tromethamine, diethanolamine andtriethanolamine of ursodeoxycholate, glycyl and taurine ursodeoxycholateand N-methyl glycyl and N-methyltaurine ursodeoxycholate.

Suitable Adhesive Polymeric Agents in Example I through V and ExampleVII are: Hydroxypropyl cellulose (klucel HF, Hercules Co.), polyvinylpyrrolidone (Plasdone, GAF Co.), a 60:40 blend of methyl cellulose andethyl cellulose (Dow Chem. Co.), Hydroxypropyl methyl cellulose (Grades50 and 55, Eastman Kodak Co.), cellulose acetate phthalate (EastmanKodak Co.) and propylene glycol alginate (Kelco Co.).

Suitable Acid-Resistant Polymers to coat the microspheres in Example Ithrough V and Example VII are: Hydroxypropyl methyl cellulose phthalate,Grades 50 and 55 (Eastman Kodak Co., or Shin-Etsu Chemical Co., Ltd.),AQUATERIC® aqueous enteric coating polymer dispersion (FMC Corp.),EUDRAGIT® acrylic based polymeric dispersion (Rohm Pharma GMBH,Germany), and cellulose acetate phthalate (Eastman Kodak Co.).

Example VIII will further illustrate the composition of theacid-resistant polymer-coating:

                  EXAMPLE VIII                                                    ______________________________________                                                               % w/w                                                  ______________________________________                                        Hydroxypropyl methyl cellulose phthalate*                                                              7.4                                                  Diethyl phthalate        2.0                                                  2-Propanol               45.2                                                 Ethylacetate             45.2                                                 Talc, USP                0.2                                                  ______________________________________                                         *When the hydroxypropyl methyl cellulose phthalate was replaced with          cellulose acetate phthalate an equally suitable acidresistant polymer         coating was obtained, as long as, talc was also included in the               composition. The presence of talc with the film forming polymer caused th     deposition of an acidimpermeable polymer coat. When AQUATERIC ® or        EUDRAGIT ® aqueous enteric coating polymer dispersion was employed in     place of cellulose acetate phthalate (CAP) or hydroxypropyl methyl            cellulose phthalate (HPMCP), the microspheres were first sealed with an       initial thin layer coating with CAP or HPMCP (2-4% w/w of the                 microspheres), followed by a secondary coating with an aqueous polymeric      latex dispersion (for example, AQUATERIC ® or EUDRAGIT ®. The         employment of the aqueous coating composition as a secondary coating is       important to reduce the evaporation of solvents into the atmosphere and       thus reduce environmental pollution.                                     

The total amount of the composition required to be administered to anenzyme/bile salt deficient patient will vary with the severity of theconditions, age and other physical characteristics of the patient. Thephysicians will prescribe the total amount, the dosage and the frequencyof dosage administration on a patient by patient basis. Generally, forenzyme/bile salt deficient patient from about 0.5 to about 1.5 grams ofthe composition are administered with each major meal, three times aday. Larger amount may, however, be required for certain conditions,such as for dissolving gallstones.

For ease of administration of the compositions it is preferred to usegelatin capsules containing about 0.2 to 0.5 grams microspheres ormicrotablets. Gelatin capsules which disintegrate in the acidicenvironment of the stomach are well-known and utilized in the prior art.Microtablets are of small size, having a diameter between about 1 to 5mm and a thickness between 0.5 to 4 mm. The microtablet is prepared byconventional tableting procedure. However, the compositions of thepresent invention in the form of very small particle sizes may be usedper se. The microspheres shown in Example IIB and IIIB (Table I) are inthe 10 to 80 mesh size range. Table I shows that 100% of themicrospheres of Example IIB were in the 20 mesh to 80 mesh size range(0.84 to 0.149 mm) and 89.7% of the coated particles IIIB were in therange of 20 to 40 mesh size range (0.84 to 0.42 mm). Young children oradults with certain diseases are unable to swallow big gelatin capsules.Microspheres of very small size of the present invention could then beadministered to the patients with liquid food, such as milk, apple sauceand semi-solid foods.

What is claimed is:
 1. A process for preparing a digestive enzyme andbile salt composition for the treatment of digestive enzyme and bilesalt deficiencies in mammals comprising the steps of:a) blending in dry,powdery form (i) about 71 to about 90% w/w of an enzyme selected fromthe group consisting of pancreatic proteases, lipases, nucleases andamylases; (ii) about 1.0 to about 61% w/w of a salt of ursodeoxycholicacid selected from the group consisting of sodium, potassium, ammonium,tromethamine, ethanolamine, diethanolamine and triethanolamine; (iii)about 0.8 to about 5.0% w/w of a buffering agent consisting of anhydroussodium carbonate, sodium bicarbonate, potassium carbonate or potassiumbicarbonate in combination with about 0.8 to about 1.5% w/w oftromethamine, diethanolamine or triethanolamine; (iv) about 0.3 to about19% w/w of an adhesive polymer selected from the group consisting ofhydroxypropyl cellulose, polyvinylpyrrolidone, cellulose acetate,phthalate and methyl cellulose; (v) about 0.9 to about 16% w/w of adisintegrant selected from the group consisting of starch, modifiedstarches, microcrystalline cellulose and propylene glycol alginate; b)wetting said blended ingredients with a liquid to cause the blend tostick together, wherein said liquid is selected from the groupconsisting of (i) 1-25% w/w ethanol, 75-99% w/w 2-propanol, and 0.2-2%w/w water, (ii) 98-99% w/w 2-propanol and 0.2-2% w/w water, and (iii)1-25% w/w methanol, 0.2-2% w/w water, 75-98% w/w 2-propanol and 1-5% w/wethylacetate; c) granulating or extruding the blend through a 10 or 18mesh S/S screen; d) converting the granules to a uniform diameterparticle size; e) compacting the uniform particles into sphericalparticles; f) drying the spherical particles; g) coating the particleswith a gastric acid-resistant polymer that disintegrates under neutralor basic conditions; and h) drying the polymer-coated sphericalparticles.
 2. The process of claim 1 wherein said digestive enzyme isselected from the group consisting of Pancreatin, Pancrelipase, Trypsin,Chymotrypsin, Chymotrypsin B, Pancreatopeptidase, Carboxypeptidase A,Carboxypeptidase B, Glycerol Ester Hydrolase, Phospholipase A2, SterolEster Hydrolase, Ribonuclease, Deoxyribonuclease, α-Amylase, Papain,Chymopapain, Bromelain, Ficin, β-Amylase, Cellulase and β-Galactosidase.3. The process of claim 1 wherein said composition is in the form ofmicrospheres having a mesh size of about 10 to
 80. 4. The process ofclaim 1 wherein said liquid for wetting the blended ingredient comprisesfrom about 0.2% to about 2.0% water.
 5. The process of claim 4 whereinsaid water content is from 0.2 to 1.5%.
 6. The process of claim 5wherein said water content is from 0.2 to 1.0%.
 7. The process of claim1 wherein the particle size range of said seeds is from about 20 toabout 60 mesh.
 8. The process of claim 1 for preparing a digestiveenzyme and bile salt composition for the treatment of digestive enzymeand bile salt deficiencies in mammals comprising the steps of:a)preparing starting seeds of the bile salt comprising blending thepowdery bile salt with the disintegrant and the buffering agent; b)spraying said blend with a solution of the adhesive polymer until theblend agglomerates; c) granulating or extruding the agglomerate througha 10 or 18 mesh S/S screen; d) converting the granules to a uniformdiameter particle size of 40 to 60 mesh; e) compacting the uniformdiameter particles into spherical particles; f) drying the sphericalparticles to obtain the starting seeds; g) tumbling the starting seedsin a rotating coating pan, wetting the starting seeds with a solution ofthe adhesive polymer and dusting the starting seeds while tumbling inthe rotating coating pan with the powdery buffered bile salt, enzyme,and disintegrant composition to obtain particles in the 10 to 20 meshsize range; h) coating the 10 to 20 mesh size particles with a gastricacid-resistant polymer that dissolves under neutral or basic conditions;and i) drying the polymer-coated particles.
 9. The process of claim 8wherein said starting seed is selected from the group consisting of (i)sodium or potassium salts or ursodeoxycholic acid, (ii) ethyl or propylesters of ursodeoxycholic acid, (iii) sodium or potassium salts ofglycyl and tauroursodeoxycholate and (iv) sodium or potassium salts ofN-methyl glycyl and N-methyl tauroursodeoxycholate.
 10. A method fortreating digestive enzyme and bile salt deficiencies in mammalscomprising orally administering to said mammals a therapeuticallyeffective amount of the composition of claim 1.